Although an HIV-1 vaccine continues to be elusive after over twenty years of effort, it remains the single best hope to stop the epidemic (1, 2). The recent failure of an Ad5-vectored ‘CTL’ vaccine and the earlier failure of a gp120 subunit vaccine are sobering testaments to the difficulty of this task (2). Because of these failures, there is renewed focus on the identification and characterization of protective humoral responses in groups of HIV-1 infected individuals who spontaneously control their infections. These groups include long-term non-progressors (LTNP) who maintain stable CD4 counts without disease over many years (3) and elite controllers (EC) (4) or, in our clinic, natural viral suppressors (NVS) (5), who control viral replication to undetectable levels without antiretroviral therapy. Several recent studies have attempted to characterize the anti-envelope antibodies (Abs) found in the plasma or sera of rare HIV-infected humans that exhibit broadly neutralizing activity (6, 7). However, the specificities of circulating Abs are likely to change and/or decline significantly over time given the high mutability of the HIV envelope (8), particularly under conditions where antigenemia is limiting. Thus, circulating Ab specificities in chronically HIV-1-infected persons are unlikely to represent the full spectrum of Abs elicited by the virus from the time of early acute infection. Further, potentially important Ab responses that occur during the critical period of acute infection might not be detected by serological analyses of samples taken after viral loads have declined to setpoint. For example, it is known that EC have lower titers of HIV-1 specific Abs than chronic progressors (9, 10). Therefore there is a need for a new approach to evaluate the present and past immune responses in HIV-infected individuals to identify new and therapeutically useful antibodies. This same issue is important in individuals that have been infected with other pathogens or who have autoimmune diseases.
Once such antibodies are identified, there is a need for a reliable method for making them, preferably as fully human antibodies for human use. Five general methods are used to isolate human monoclonal antibodies (mAbs). Each method suffers from technical limitations that render it difficult to use in comparison with the original Kohler-Milstein hybridoma method used to isolate murine mAbs. These methods include hybridomas (40), B cell transformation with Epstein-Barr virus (EBV) (41), phage display (42), yeast display (43), and ‘humanization’ of murine mAbs (44). Due to the lack of a generic cell fusion partner, it has proven difficult to reproducibly isolate human mAbs by conventional hybridoma methods, although this method has proven successful (c.f, for an interesting recent example (45)). EBV transformation has proven equally difficult but for different reasons. EBV transformed B cell lines are often genetically unstable and the consequent high rate of clone loss makes this method very cumbersome.
Recently, a modified EBV method was described that uses EBV to transform enriched memory B cells (BMem) from immunized BMem (26). This method works well for many immunogens but it requires normal BMem, which are often lacking during chronic infections such as HIV-1 (18). Phage and yeast display overcome these problems but these methods rely on either Fab fragments or single-chain Fv (scFv) fragments in the screening obviating the uses of these methods for screening by functional analyses such as viral neutralization. Phage display also suffers from the problem that it imposes unidentified structural restrictions on antibody specificities that can be stably expressed as recombinant phage (46). Finally, it is possible to ‘humanize’ murine mAbs by grafting the gene segments that encode antigen binding onto human variable region genes. This is a highly labor intensive endeavor and beyond the reach of most research laboratories. Therefore there is a need for a new method to rapidly clone full-length human mAbs that obviates these problems.